Production of riboflavin



Patented mm, 1949 PRODUCTION OF RIBOFLAVIN Allen S. Phelps, Orangeburg,N. Y., assignor, by mesne assignments, to American Cyanamid Company, NewYork, N. Y., a corporation of Maine No Drawing. Application December 11,1945, Serial No. 634,395

2 Claims.

This invention relates to an improvement in the art of producingriboflavin. More particularly, the present invention is concerned withimprovements in the method in which the fungus Eremothecium ashbyii isused to produce ribo-' fiavin by fermentation of a liquid, nutrientmedium. The invention includes the fermentation process as well as thenew medium.

In the co-pending application of Henry D. Piersma, Serial No. 505,189,filed October 6, 1943, now United States Patent 2,400,710, issued May21, 1946, a process is described and claimed by which good yields ofriboflavin are produced in a liquid medium comprising water, an animalproteinaceous substance, malt extract, and sugar by the action of theorganism Eremothecium ashbyii. By this process yields of riboflavin ofthe order of 200 gammas per milliliter are ob tained. Occasionally, in alaboratory experiment, the yields of riboflavin are somewhat higher.

Although one of the essential features of novelty of the Piersma processis the use of malt extract,-the use of which leads to much higher thannormal yields of riboflavin, the presence of animal proteinaceousmaterial is a critical and necessary part of the fermentation liquor.Although the process of the Piersma application just mentioned disclosesthe use of a wide variety of animal proteinaceous materials, theparticular products described are all derived from animal tissue. Thismaterial is water-insoluble, is generally non-homogeneous in chemicalcomposition.

and physical form and is frequently obtained in a contaminated conditionwith bacterial decomposition in a well advanced state. In short, animaltissue is not the most desirable substance that could be used in aprocess which is easily contaminated with foreign organisms which causegreatly diminished yields of riboflavin.

The process of the co-pending application of.

Henry D. Piersma, Serial No. 572,747, filed January 13, 1945, representsa considerable improve- 2 enzymatic treatment it is possible to obtaingreatly increased yields of riboflavin, generally on the order of 400gammas per milliliter although, as might be expected, individuallaboratory experiments sometimes show higher yields of riboflavin.

This second named process has been used for the commercial production ofriboflavin with very good results. It is subject, however, to theearlier mentioned disadvantages caused by the use of animal tissue. Itis also subject to the disadvantage of requiring an enzymatic digestion,which adds time and expense to the process. Although some of the animaltissue is solubilized by the enzymes, most of it remains insoluble andmust be filtered from the liquor at the end of the fermentation process.Also, as in the case of the undigested animal tissue, and because of thefact that the fermentation is usually conducted at substantialneutrality, the medium is subject to contamination by organisms whichinterfere with the production of riboflavin.

In view of the disadvantages of the use of water-insoluble animalproteinaceous material in the riboflavin process, it is one of theobjects of the present invention to provide an improved fermentationmedium which eliminates or minimizes the disadvantageous features ofmedia formerly employed. One of the objects of the present invention is,therefore, to prepare a liquid fermentation liquor in which all, orpractically all, of the nutrient medium is in solution thus making itpossible to sterilize the liquor, filter it, and otherwise handle itwith greater facility. Such a liquor is also less apt to becomecontaminated during the fermentation process. Itis also an object of theinvention to eliminate the expensive enzyme digestion treatment, whichis necessary for best results when using animal tissue of the typeheretofore employed. Still another object of the present invention is toprovide a fermentation liquor which is simpler to prepare and easier tohandle in the plant. Other objects of the invention are to provide afermentation liquor which is more homogeneous than those hithertoavailable, and to make it possible to shorten the cooking cycle, andgenerally inapparent hereinafter, are accomplished by me by the use of aliquid medium for the production of riboflavin by the action of thefungus Eremotheciumashbyii which comprises water, a sugar, malt extract,and milk or a milk product containing solubilized casein. The use ofcasein as the animal proteinaceous substance eliminates most of thedisadvantages of the water-insoluble animal tissue previously employedand at the same time results in greatly increased yields of riboflavin.As will be seen from the examples which will be described hereinafter,yields of riboflavin between 500 and 600 gammas per milliliter areusually obtainable when using the improved medium of the presentinvention. As the casein is used in a solubilized form, the medium canbe quickly and easily prepared, sterilized, pumped, filtered, andotherwise handled in the plant with a minimum of effort. Because of theabsence of large particles of solid material it is easier to completelysterilize it and there is less danger of contamination by harmfulorganisms. As will be apparent, enzymatic digestion is avoided, thuslowering the cost of the process and shortening the time required forprocessing the material.

The casein which is used may be the ordinary casein of commerce, whichis prepared by treating milk with an acid to precipitate casein whichisthen washed and dried. In using this material it is first solubilizedby treatment with an alkali, such as ammonia, caustic soda, sodiumcarbonate, etc. in a manner known to those in the art. Instead of usingcommercial dried casein I may employ freshly precipitated casein or, ifavailable, casein containing products in which the casein is alreadysolubilized such as whole milk, skim milk, fresh milk, sour milk, buttermilk, evaporated milk, and the like. Other milk products which contain asubstantial amount of casein and which may be solubilized may also beused. The term solution as used includes colloidal solutions in whichthe casein does not settle out on standing. The concentration of themilk product in the fermentation liquor is adjusted on the basis of thecasein content to a level equivalent to about 0.4 to 4.0% by weight ofcasein (dry basis) based on the total weight of the liquid medium. Thepreferred range is around 0.8 to 1.0%.

The malt extract employed in the medium is an article of commerce andconsists essentially of a concentrated, aqueous extract of malted grain,such as barley. As will appear obvious, ground malt itself may be addedto the mash in lieu of malt extract but such material is not ordinarilyemployed as it adds insoluble substances to the fermentation liquor. Thepresence of hop extracts, which are sometimes found in commercial maltextracts, are not deleterious and may, in fact, be advantageous adjunctsfor the growth media. The amount of malt extract used in the medium maybe varied from about 0.2% to 30% dry basis which is equivalent to about0.5 to 5.0% by weight as received, the optimum amount appearing to befrom about 0.7 to 1.0% dry basis.

The sugar used in the medium is preferably glucose or one of the invertsugars or sugar syrups of commerce. Other sugar, such as sucrose,fructose, or mannose, lactose and galactose may also be used to supplythe carbohydrate requirements of the medium. The term sugar as usedhereinafter and in'the claims does not include the maltose or dextims ofthe salt extract. The amount of sugar in the medium is ordinarily withinthe range of 0.2% to 4.0% by weight, based on the total weight of themedium. The preferred range is about 0.3% to about 0.6%.

As previously indicated, the riboflavin-producing organism of theprocess is the fungus Eremothecium ashbyii. The fermentation of thenutrient liquor with this organism is carried out within the range ofabout 20 C. to 35 0., the preferred range being from about 30 to 32 C.The fermentation process is conducted at substantial neutrality, the pHof the mash being adjusted to within the range of 6.0 to 8.5 beforesterilizing. Fermentation is preferably carried out between pH 6.5 andpH 7.5.

Inasmuch as the fungus Eremothecium ashbyii requires oxygen forsatisfactory growth and production of riboflavin, it is necessary toaerate the medium during the fermentation process. This may beaccomplished by growing the organism with agitation in an open vesselcontaining a thin layer of medium. In such a process the ratio ofsurface of medium exposed to the atmosphere in square centimeters to thevolume of'the medium in cubic centimeters should be at least 1.0.

A more practical way of fermenting large volumes of the liquor is toadopt the conventional practice of blowing a finely dispersed stream ofair through the liquor contained in a tank. Inasmuch as this method ofsupplying air to aerobic organisms in deep tank cultures has beenpracticed for many years by the fermentation industry, detaileddiscussion would appear to be unnecessary. In general, however, thediffusers should have the smallest openings practical so that the air isfinely dispersed through the medium. The air flow should be at least0.25 cubic foot per minute per square foot per surfac of the mash.Larger amounts of air are more desirable, preferably from about 1 to 2cubic feet per minute per square foot of liquid surface. Under somecircumstances mechanical agitation may be desired but ordinarily theagitation of the liquor caused by aeration will be sufficient.

The progress of the fermentation can be readily ascertained bywithdrawal of samples from the fermentation vessel. The duration of theactive phase of the fermentation is subject to considerable variationand may be as short as 60 hours or as long as 140 hours. Best resultshave been obtained in plant practice at around to hours.

To illustrate the invention in greater detail the following examples aregiven. These examples are based on laboratory experiments but analogousresults have been obtained by large scale fermentations in tankscontaining several thousand gallons of medium.

EXAMPLE 1 A series of media containin Various milk products wasprepared. These media each contained 1.75% of a commercial malt extract(weight as received, 50-60% solids), 0.5% of Cerelose (glucose hydrate,dry weight), and the indicated amount of milk product. The media wereadjusted to a pH between 6.5 and 6.9 before sterilization. A quantity ofthe media, 25 ml. portions, was placed in 250 ml. Erlenmeyer flasks,sterilized at 15 pounds steam pressure for 30 minutes and inoculatedwith a culture of Eremothecium ashbyii. Incubation was carried out at 30to 32 C. with the flasks in a shakin machine which completedoscillations per minute for a period of 88 hours. The results of thisseries of experiments are shown in the following table. The ammoniatreated casein was a commercial brand of casein which had beensolubilized by treatment at 45 to 50 C. in solution with 0.? ml. of 26%ammmonium hydroxide for each 10 grams of casein.

' TABLI 1 Riboflavin yield from various milk products Average -Mill:Product Amount Riboflavin Yield Wh milk 8 oo 605 Row Skim Milk 8 co 650Dry Skim Milk. 0.8 gm- 610 Condensed Milk- 4 cc 070 Evaporated Milk- 4cc. e90 Ammonia treated dried casein 0.25 gm--- 580 I 1 Quantity usedper 26 ml. of medium.

As will be seen from the foregoing results, very excellent yields ofriboflavin are obtained when using casein from a variety of milkproducts.

.EXAMPLE2 To illustrate the fact that casein is the essential factor ofthe milk products employed a second series of experiments was carriedout with diflerent milk fractions. To obtain these fractions raw skimmilk was adjusted to a pH of 4.7 with sulfuric acid and heated to 55 C.The resulting curd was filtered off and washed twice by suspending inwater and buffered in water at a pH oi 4.7 with a small quantity ofsodium acetate. This washed precipitate dissolved by stirring withexcess alkali and adjusted to a pH between 6.5 and 6.8 is referred to ascasein. The combined filtrates from the above treatment were adjusted to6.5, heated to 100 0., filtered hot, and the precipitate washed with hotwater. This precipitate, dissolved by stirring with excess alkali andadjusted to 6.5 to 6.8, is referred to as albumin." The final combinedfiltrates from the above steps are referred to as "whey. Preparation,steriliza-' Average Riboflavin Yield, 1,111].

$3; Whey Casein Albumen NNNN NNNN

I All fractions were added to each flaskin the proportions present in 8cc. voi raw skim milk.

These results clearly demonstrate that casein is the factor in milkwhich leads to the high yields of riboflavin.

EXAMPLE 3 To demonstrate the fact that casein alone is insumcient toresult in high yields of riboflavin another series of experiments wasconducted. In this series the basal medium was varied by eliminatingcertain of the nutrient elements as indicated. Otherwise the conditionsof the fermentation were as in the preceding examples. The casein usedwas a commercial grade ofcasein which had been solubiiized by treatmentwith ammonium hydroxide as in Example The results were as follows:

The first three entries in the table show that none of casein, maltextract or Cerelose when used alone will allow the Eremothecium ashbyiito produce any significant amount of riboflavin. when malt extract andCerelose are used together with the omission of casein only slightlyimproved yields of ribflavin are obtained. The table also shows thatwhen either malt extract is omitted or when the sugar is omitted, theyields of riboflavin are not satisfactory. However, when all threeessential components are present, very good yields of riboflavin areobtained.

These results demonstrate the fact that the improved results obtained bythe present invention are not entirely dependent upon any one factor butare the results of the combined effects of the three essentialcomponents of the medium: casein, malt extract, and sugar, insubstantially the proportions indicated. As previously noted, however,some variation is permissible in these proportions and, in fact, betterresults may be obtained when the various components are in slightlydifferent relationship to each other.

Optimum conditions are easily determined by' those skilled in the artwith a small amount of experimentation.

I claim:

1. A proces of producing riboflavin which comprises growing the fungusEremothecium ashbyii in an aqueous medium containing in solution theessential components water, about 0.4 to 4.0% by weight of casein, fromabout 0.2 to 3.0% by weight of malt extract and, from 0.2 to 4.0% byweight of sugar while aerating the medium.

2. A process of producing riboflavin which comprises the steps ofinoculating an aqueous medium containing in solution the essentialcomponents water, about 0.4 to 4.0% by weight of casein, from about 0.2to 3.0% of malt extract, and from 0.2 to 4.0% of glucose with the fungusEremothectum ashbyii and allowing fermentation to take place at atemperature between about 20 to 35 C. for a period of time between 60and hours while aerating the medium.

' ALLEN S. PHELPS.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS OTHER REFERENCES Rally: Comptes Rendus, vol. 209(1939), pages 900 902.

